HCV核心区多肽对细胞毒T细胞作用的初步观察

目的：探讨HCV感染者体内细胞毒T细胞（CTL）功能缺陷的原因，为深入研究HCV感染的发病机制和指导疫苗研制提供理论和实验依据。方法：采用多肽固相合成法选择性合成HCV核心区多肽，并将皮下注射免疫Balb/c小鼠，用乳酸脱氢酶（LDH）释放实验检测小鼠脾细胞（CTL）活性。结果：经单因素方差分析显示，HCV核心区多肽CPA9（39－74位氨基酸），CPB7（67－76位氨基酸），CPB8（71－80位氨基酸）对小鼠CTL有抑制作用，CPA10（5－23位氨基酸），CPB6（63－72位氨基酸）和CPB2（131－140位氨基酸）对小鼠CTL有增强作用，结论：经初步观察，HCV核心区多肽对CTL有抑制作用及增强作用。     

丙型肝炎病毒特异性细胞毒性T淋巴细胞活性研究

目的阐明丙型肝炎病毒(HCV)特异性细胞毒性T淋巴细胞(CTL)在慢性丙型肝炎病毒感染中的作用.方法用标准铬释放法(以从患者肝组织或外周血单个核细胞中经选择性克隆扩增后的CD8+细胞为效应细胞,经EB病毒转染的自身B淋巴母细胞为靶细胞,由能表达HCV1型核心区基因的重组痘苗病毒作为转导载体)对62例慢性丙型肝炎患者肝组织及外周血单个核细胞(PBMC)中的HCV特异性CTL活性(HCV CTL)进行检测,8例非HCV感染的肝病患者作为对照.结果62例慢性丙型肝炎患者中,共有28例(46 7%)肝组织中检测出HCV CTL活性,但HCV-CTL在PBMC中未检出.对照组患者肝组织及PBMC中均未检出.5例非HCV1感染的丙型肝炎患者检测出针对HCV1型表位的HCV-CTL.HCV-CTL阳性的丙型肝炎患者血清丙氨酸转氨酶(ALT)、天门冬氨酸转氨酶(AST)水平及肝组织活动指数均明显高于HCV-CTL阴性的患者,而其血清HCVRNA水平则显著低于后者(P＜0.01).结论1 HCV-CTL主要存在于肝组织内;2存在型交叉性HCV-CTL;3.HCV-CTL活性阳性的患者较HCV-CTL活性阴性者具有较高的疾病活动度及较低的病毒血症水平;4.HCV特异性CTL在丙型肝炎的发病机制及疾病控制中起重要作用.     

丙型肝炎病毒核心区蛋白和细胞凋亡对细胞端粒酶活性的影响

丙型肝炎病毒(HCV)核心区编码的HCV-C蛋白维持病毒外形，还有较强的免疫调节功能，是细胞免疫的主要识别位点，在HCV致病过程中起着重要作用。端粒酶活性上调被认为是肿瘤发生的一种机制，HCV引发肝细胞癌(HCC)是否与上调肝细胞端粒酶活性有关?本实验拟对上述问题进行初步探讨，以部分阐明HCV导致HCC发生的机制。     

丙型肝炎病毒特异性细胞毒性T淋巴细胞功能的研究

目的 研究慢性丙型肝炎患者丙型肝炎病毒(HCV)特异性细胞毒性T淋巴细胞(CTL)的功能及其与临床疾病状态的关系.方法 表达HCV核心蛋白的真核表达质粒pcDNA3.1-core通过Lipofecta-mineTM基因转染法转染HepG2细胞,经G418筛选获得稳定转染HepG2细胞(Hep-core),经Western blot证实有HCV核心蛋白表达;分离患者外周血单个核细胞,经体外诱导扩增获得HCV特异性CTL(HCV-CTL),以Hep-Core细胞和HepG2细胞作靶细胞,乳酸脱氢酶释放法检测HCV-CTL活性,用酶联免疫吸附法检测培养上清液中干扰素-γ(IFN-γ)含量,以正常人作为对照.结果 慢性丙型肝炎患者组HCV-CTL活性值为(23.9±4.8)%,明显低于对照组(42.6±6.5)%(t=7.22,P=0.011).高病毒载量组HCV-CTL活性值(18.9±4.8)%,明显低于低病毒载量组的(33.7±3.2)%(t:8.22,P=0.003);基因1型患者HCV-CTL活性值(20.8±2.1)%明显低于基因2或3型的(32.4±2.5)%(t=11.7,P=0.001);ALT升高患者HCV-CTL活性值(29.3±3.1)%与ALT正常患者(25.7±3.4)%相比无统计学差异(t=0.93,P＞0.05).慢性丙型肝炎患者培养上清液中的IFN-γ量(957±241)pg/ml明显低于对照组的(3117±673)pg/ml(t=8.87,P=0.001).结论 慢性丙型肝炎患者HCV-CTL活性降低,CTL的免疫功能低下与病毒水平和基因型相关而与ALT水平无关.     

慢性肝炎患者血清中丙型肝炎病毒核心区和包膜1区基因变异研究

目的 探讨丙型肝炎病毒（HCV）核心（C）区和包膜1（E1）区变异与其感染慢性化的关系。方法 10例HCV慢性感染者和2例急性感染者血标本,采用逆转录－聚合酶链反应（RT－PCR）扩增HCV的C区羧基端、E1及E2区氨基端片段（1kb),扩增产物进行克隆,以单链构象多态性（SSCP）和异质性双体（HD）分析对每份血清的30个克隆的C／E1区准种（quasispecies)进行筛选,挑选每例标本HCV的优势株与劣势株序列进行测定,并分析推导的氨基酸序列。结果 各例患者血清中HCV的C／E1区扩增片段形成的SSCP条带间差异明显,而HD与同源双体之间差距不明显。HCV急性感染者和慢性感染者血清病毒C区无发生变异,后者E1区氨基酸替换率为1．32％,但功能性氨基酸无改变。结论 HCV的E1区序列变异以准种形式存在,但与免疫逃避可能无关。     

乙型和丙型肝炎病毒对主要组织相容性复合体基因的调节

众所周知，免疫学上把能引起强而迅速排斥反应者称为主要组织相容性抗原，其编码基因是一组紧密连锁的基因群，称之为主要组织相容性复合体(major histocompatibility complex，MHC)。MHC在不同的哺乳动物中有不同的命名，但是其组成、结构以及编码产物的功能等却很相似。人主要组织相容性抗原称为人白细胞抗原系统(human leucocyte antigen，HLA)。     

丙型肝炎病毒对新生多肽相关复合物α多肽基因的表达调节研究进展

新生多肽相关复合体 (NAC ,nascentpolypeptide as sociatedcomplex)是一种由α和 β多肽组成的异二聚体复合物 (heterodimericcomplex) ,可逆性地与真核细胞核糖体结合 ,位于核糖体新合成多肽的顶端 ,引导新合成的多肽在细胞内的正确分布与转位[1] 。尽管从低等的酵母到高等的哺乳动物细胞都有这种基因表达的存在 ,但其生物学功能至今还不完全清楚。研究结果表明 ,NAC的生物学功能可能包括 :第一 ,为新生多肽合成、分泌提供动态变化的核糖体外出通道 ;第二 ,进入内织网 (ER)细胞内转位的负调节因子 ;第三 ,进入线粒体的正调节因子。但…     

中国丙型肝炎病毒细胞毒性T淋巴细胞表位预测及其多表位疫苗设计

寻求抗HCV疫苗和有效抗病毒药物的探索一直是研究的热点[1].我们应用生物信息学方法,选择HCV核心(C)区、NS3、NS4、NS5等各区的细胞毒性T淋巴细胞(CTL)表位,进行优势组合,结合中国人人类白细胞抗原(HLA)的频率,设计、合成重组HCV多表位疫苗的基困序列,以达到对CTL表位初步筛选,提高实验成功率的目的,同时为构建多个CTL表位疫苗提供理论依据,为今后HCV疫苗的研究开发积累一定的实践经验.     

丙型肝炎病毒多肽C1,C2,N1·N2合成及其应用

根据丙型肝炎病毒（ＨＣＶ）核苷酸和氨基酸序列合成了ＨＣＶ结构区核心部位Ｃ１、Ｃ２和非结构区Ｎ１·Ｎ２三个多肽。建立了一个敏感和特异的酶联免疫吸附试验（ＥＬＩＳＡ）检测抗－ＨＣＶ，与美国抗－ＨＣＶ第２代试剂盒比较，符合率为９７．７％。检测临床标本１０２５份，抗－ＨＣＶ阳性率在慢性非甲非乙型肝炎（ＮＡＮＢＨ）４３．９％，血透患者３４．４％，肝硬化２５．０％，急性ＮＡＮＢＨ１７．４％，肝癌１６．７％，助     

丙型肝炎病毒细胞模型和动物模型的研究

建立丙型肝炎病毒(HCV)的细胞模型及动物模型,对于深入认识HCV病原学特点、复制及发病机制,筛选有效药物及疫苗等极为重要。近年来,国内外学者作了大量探讨,概括如下。 1 HCV细胞模型的研究 1.1 HCV感染类细胞模型 1.1.1 以肝细胞为宿主的HCV感染细胞模型 众所周知,人肝细胞是HCV感染的天然宿主细胞,以成人肝细胞作为感染HCV的细胞模型应有更好的代表性。Ito等直接将HCV阳性血清与丙肝患者肝活检分离出来的肝细胞共培养,发现肝细胞内和培养上清液中可检出大量HCV RNA,用     

树突状细胞的非特异性免疫功能降低对HBV、HCV清除及细胞毒性T淋巴细胞应答无影响

目的 观察HBV、HCV感染者树突状细胞(DC)的非病毒特异性免疫功能状态与细胞毒性T淋巴细胞(CTL)免疫应答以及病毒清除的关系。方法 对25例成人慢性HBV和HCV合并感染者进行了间隔8年的两次调查、依据临床转归分为HBV和HCV均清除组(A组)14例、单独HCV清除者(B组)6例，单独HBV消除者(C组)3例，HBV和HCV均未清除者(D组)2例，对照组(N组)为同一地区健康献血员11例。体外分离培养DC，检测其表型及抗原摄取功能、刺激异体淋巴细咆增殖能力和4组感染者的CTL免疫应答情况。结果 B、C、D组与A组、N组比较，DC的非病毒特异性免疫功能降低，表现为CD86表达的降低、刺激异体淋巴细胞增殖的能力下降以及抗原摄取能力降低。A组对HBV和HCV的4条抗原表位多肽均有较高的CTL应签率(11／12)；B组对HCV的两条抗原表位多肽均有应答(5／5)，但无对HBV两条表位多肽均应签者、仅有1例对P2有反应；C组对HBV的抗原表位多肽均有应答，但无对两条HCV表位多肽均应答者；D组及N组对HBV或HCV所有实验多肽均无应答。结论 HBV和HCV的清除与病毒特异性的CTL应答相关。HBV和(或)HCV持续存在可能是导致DC功能异常的原因。     

人类免疫缺陷病毒和丙型肝炎病毒重叠感染者的临床及免疫特征

目的 观察人类免疫缺陷病毒(HIV)和HCV重叠感染者与慢性丙型肝炎患者临床特征及HCV特异性细胞毒性T淋巴细胞(CTL)的数量及功能,探讨两组患者免疫功能的差异及其可能的影响因素.方法 以HIV和HCV重叠感染患者59例、慢性丙型肝炎患者36例为研究对象,取治疗前外周血检测肝脏生物化学指标、血常规、外周血T淋巴细胞亚群(CD4+T、CD8+T淋巴细胞计数)及HIV、HCV病毒载量,以酶联免疫斑点法检测HCV特异性CTL的数量和功能,统计学分析两组问免疫功能的差异及与上述检测指标的相关性. 结果 中国河南省有偿献血、单采血浆人群HIV感染者中HIV和HCV重叠感染率达60.8%.ALT、AST值在重叠感染组与HCV组间差异无统计学意义;球蛋白在重叠感染组为(40.3±5.8)g/L,HCV组为(32.8±6.3)g/L,差异有统计学意义(P＜0.01).重叠感染组外周血CD4+T淋巴细胞数明显低于HCV组(P＜0.01),而CD8+T淋巴细胞数高于HCV组(P＜0.01).重叠感染组HCV RNA定量高于HCV组(P＜0.01).重叠感染组对HCV-NS3区肽段的反应强度(每106个外周血单个核淋巴细胞中斑点形成细胞的个数)较HCV组弱,649.34±685.90对比1233.70±1085.16,差异有统计学意义(P＜0.05).重叠感染组白蛋白与HCV病毒载量呈现负相关(r=0.540);重叠感染组对HCV-NS3区肽段反应强度与HIV病毒载量负相关(r=0.356);重叠感染患者CD4+T淋巴细胞数与血小板正相关(P＜0.05).但未见重叠感染组HCV RNA与CD4+T淋巴细胞数量及HIVRNA水平有相关关系.结论 重叠HIV感染有利于HCV的复制,而HIV载量可影响针对HCV的特异性免疫反应,HIV载量高则不利于HCV的清除.慢性丙型肝炎患者重叠HIV感染时,病情易慢性化,预后更差.     

Distinct Toll-like receptor expression in monocytes and T cells in chronic HCV infection

AIM: Hepatitis C virus often establishes chronic infections. Recent studies suggest that viral and bacterial infections are more common in HCV-infected patients compared to controls. Pathogens are recognized by Toll-like receptors (TLRs) to shape adaptive and innate immune responses. METHODS: In this study, to assess the ability of HCV-infected host to recognize invading pathogens, we investigated Toll-like receptor expression in innate (monocytes) and adaptive (T cells) immune cells by real-time PCR. RESULTS: We determined that RNA levels for TLRs 2, 6. 7, 8, 9 and 10 mRNA levels were upregulated in both monocytes and T cells in HCV-infected patients compared to controls. TLR4 was only upregulated in T lymphocytes, while TLR5 was selectively increased in monocytes of HCV-infected patients. MD-2, a TLR4 co-receptor, was increased in patients' monocytes and T cells while CD14 and MyD88 were increased only in monocytes. CONCLUSION: Our data reveal novel details on TLR expression that likely relates to innate recognition of pathogens and immune defense in HCV-infected individuals.     

Distinct toil-like receptor expression in monocytes and T cells in chronic HCV infection

AIM: Hepatitis C virus often establishes chronic infections. Recent studies suggest that viral and bacterial infections are more common in HCV-infected patients compared to controls. Pathogens are recognized by Toil-like receptors (TLRs) to shape adaptive and innate immune responses.METHODS: In this study, to assess the ability of HCV-infected host to recognize invading pathogens, we investigated Toil-like receptor expression in innate (monocytes) and adaptive (T cells) immune cells by real-time PCR.RESULTS: We determined that RNA levels for TLRs 2, 6. 7, 8, 9 and 10 mRNA levels were upregulated in both monocytes and T cells in HCV-infected patients compared to controls. TLR4 was only upregulated in T lymphocytes, while TLR5 was selectively increased in monocytes of HCV-infected patients. MD-2, a TLR4 coreceptor, was increased in patients' monocytes and T cells while CD14 and MyD88 were increased only in monocytes.CONCLUSION: Our data reveal novel details on TLR expression that likely relates to innate recognition of pathogens and immune defense in HCV-infected individuals.     

Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes

AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide "ALAHGVRAL (core 150-158)". The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.     

Induction of hepatitis C virus-specific cytotoxic T and B cell responses by dendritic cells expressing a modified antigen targeting receptor

AIM: To find a novel antigen (Ag) presentation strategy to improve the immune responses induced by dendritic cell (DC) vaccine expressing hepatitis C virus (HCV) core antigen (pcDNA3HCV C-Fc) in Balb/c mice (H-2d). METHODS: pcDNA3HCV C-Fc plasmid and eukaryotic expression vector pcDNA3 were injected into mice sc. Immune responses to pcDNA3HCV C-Fc were studied. Meanwhile the effect of pcDNA3HCV C-Fc on anti-translated subcutaneous tumor of SP2/0 cells stably expressing HCV C Ag (SP2/0-HCV C-FC) was also studied. Anti-HCV C in serum was detected by enzyme-linked immunoadsordent assay (ELISA) and HCV specific cytotoxic T lymphocyte (CTL) activity was measured by LDH release assay. After 3 wk of DNA immunization, the cells of SP2/0-HCV C-FC were inoculated into mice subcutaneously and tumor growth was measured every 5 d. The survival rate and living time of mice were also calculated. RESULTS: After 4 wk of DC immunization, the A450 nm values of sera in mice immunized with pcDNA3HCV C-Fc-DC and pcDNA3-DC were 0.56±0.17 and 0.12±0.03 respectively. The antibody titres in mice codeliveried with pcDNA3HCV C-Fc with DC were significantly higher than those of mice injected with pcDNA3-DC. The HCV specific CTL activities in mice coinjected with DC and pcDNA3HCV C-Fc or empty expression vectors were(73.2±3.1) % and (24.4±8.8) % , which were significantly higher than those of mice injected with water. The DC vaccine could evidently inhibit tumor growth, prolong the survival time of mice and improve the survival rate of mice and these effects could be improved by HCV C-Fc (pcDNA3HCV C-Fc) gene codelivered. CONCLUSION: DC vaccine has a strong antigenicity in humoral and cellular immunities, which can be promoted by transduced pcDNA3HCV C-Fc expressing HCV C or Fc. Thus, pcDNA3HCV C-Fc-transduced DCs may be a promising candidate for a CTL-based vaccine against HCV.     

乙型肝炎患者HBV X蛋白特异性CTL表位变异分析

目的 比较急性乙型肝炎、慢性乙型肝炎与慢性重型乙型肝炎HBV X 蛋白特异性CTL表位变异差异,探讨乙型肝炎重症化和慢性化的可能机理.方法 对393例乙型肝炎患者的血清样本进行HLA-A2分型;用巢式PCR扩增血清HBV前S/S基因与X基因并对PCR产物进行序列测定;根据HBV前S/S基因序列,用VirusBlast软件鉴定患者感染的HBV基因型;用Vector NTI软件对目前已知的6个HLA-A2限制性X蛋白特异性CTL表位序列分析,对患者各个表位变异的差异进行卡方检验.结果 190例(48.35%)患者HLA-A2阳性,其中急性乙型肝炎(AHB)67例,慢性乙型肝炎(CHB)52例,慢性重型乙型肝炎(CSHB)71例.CTL表位变异分析结果如下:对三组所有患者进行比较时,X92-100表位变异发生频数三组患者比较有非常显著性差异(P＜0.01);对三组中HBV C基因型患者进行比较时,X92-100和X115-123表位变异发生频数三组患者比较有显著性差异(P＜0.05);对三组中HBV B基因型患者进行比较时,各表位变异发生频数三组患者比较无显著差异.结论 某些HBV X蛋白特异性CTL表位在AHB、CHB和CSHB患者间变异有明显差异且受病毒基因型影响,CTL表位变异可能与乙型肝炎的重症化和慢性化机制相关.     

A novel cytotoxic T-cell epitope presented by HLA-A24 molecule in hepatitis C virus infection

BACKGROUND/AIMS: It has been suggested that cytotoxic T lymphocytes (CTL) have crucial roles for the hepatocellular damage in hepatitis C virus (HCV) infection. A series of CTL epitopes located in the HCV protein have been identified. However, no CTL epitopes restricted by HLA-A24, a common HLA allele in humans, has been identified. METHODS: Peripheral blood and liver infiltrating mononuclear cells from the patients with hepatitis C virus infection and healthy controls were stimulated with a series of peptides containing HLA-A24 binding motifs located in HCV protein. RESULTS: An immunodominant HLA-A24 restricted CTL epitope (A24-4; AYSQQTRGL, amino acids 1031-1039) presented by HLA-A24 molecule was identified using a series of synthetic peptides containing the HLA-A24 binding motifs. The CTL activity against this peptide was induced both in peripheral blood and liver infiltrating mononuclear cells from HLA-A24-positive chronic hepatitis C patients, not from HLA-A24-negative patients and HLA-A24-positive healthy controls. CTL activity was blocked by anti-HLA-A24 and anti-CD8 antibodies, not by anti-CD4 antibody. Furthermore, the A24-4-specific CTL recognized the HCV gene transfected target cells. CONCLUSIONS: Because this peptide is presented by a common HLA class I molecule, it might be useful for protection against hepatocellular damage and vaccine development in large population of the HCV-infected patients.     

树突状细胞HBsAg疫苗抗乙型肝炎病毒免疫的体外研究

目的 研究人单核细胞来源的树突状细胞（DC）激活的HBsAg特异性细胞毒性T淋巴细胞（CTL）对表达HBsAg的HepG2/S靶细胞的杀伤效应，以探索DC-HBsAg疫苗在抗乙型肝炎病毒（HBV）中的作用。方法 从健康外周血中分离邮单核细胞，在粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素4（IL-4）的作用下培养7d诱导出DC，然后以DC为刺激细胞在体外诱导出HBsAg特异性CTL；将携有HBV-S基因的pLXSN/S重组质粒电击导入肝癌细胞系（HepG2)，建立表达HBsAg的靶细胞模型HepG2/S；用染色法检测HBsAg特异性CTL对HepG2/S靶细胞的杀伤效应。结果 DC激活的HBsAg特异性CTL对HepG2/S靶细胞具有较强的杀伤效应，不同浓度HBsAg(0μg/L，50μg/L和100μg/L）诱导的CTL的杀伤率分别为3.8%,69.5%和85.1%，而CTL对HepG2细胞无明显杀伤效应。结论 由DC激活的HBsAg特异性CTL具有较强制 特异性抗HBV作用。